One of the most important infectious causes of morbidity and mortality world wide is M. tuberculosis the causative agent of tuberculosis. There has been a global increase in the incidence of tuberculosis, as well as an increase in the United States, due to the spread of HIV infection. In Asia, the combination of HIV and tuberculosis is already considered by many to be the most significant infectious disease problem. In India, which has a huge reservoir of infected persons, tuberculosis is already the major cause of death in HIV infected individuals. The long history of research into the pathogenesis of tuberculosis has provided ample evidence that the ability of M. tuberculosis and other pathogenic mycobacteria to survive and replicate within the host macrophage is a crucial factor in pathogenesis. The goal of our research is to identify mycobacterial genes which confer upon M. tuberculosis the ability to successfully manipulate the host immune system to its own advantage. The starting point for research is to study the interaction of pathogenic Mycobacteria with the host cell, to characterize cellular and bacterial components required for bacterial entry into and survival within host cells, and to identify bacterial components required for long term survival in the host. Mycobacteria species which are pathogenic for immunocompetent humans include M. tuberculosis, M. bovis, M. leprae, M. marinum and M. ulcerans. Studies in our laboratory have shown that M. marinum and M. ulcerans are more closely related to M. tuberculosis than any Mycobacterial species except for M. bovis. Our initial studies have been on the use of M. marinum as a model for studying bacterial-host cell interactions. We have determined that like M. tuberculosis, M. marinum enters both phagocytes and non-professional phagocyte cell lines. In addition, M. marinum r, like M. tuberculosis replicates within and is cytotoxic for macrophage cell lines. In order to more carefully assess the intracellular phenotype of M. marinum studies were undertaken to characterize the intracellular compartment within which M. marinum survive. These studies shown that M. marinum, like M. tuberculosis , resides in a unique endosomal vesicle which does not fuse with the lysosome. This failure to fuse with the lysosome, enables bacteria to survive within the normally lethal environment of the macrophage. We have developed a novel strategy for looking at the expression of mycobacterial genes within the macrophage. Preliminary results from these studies show that macrophage -induced M. marinum genes have homology to genes found in M. tuberculosis. The results of these studies can be expected to provide considerable insight into how M. tuberculosis causes disease. Such information may lead not only to the identification of novel targets for therapeutic drugs, but may also provide insight into the fundamental processes by which eucaryotic trafficking pathways function. Like M. tuberculosis, M. ulcerans causes a persistent infection in humans, Buruli ucler. The pathogenesis of this disease includes extreme tissue necrosis in the absence of an acute inflammatory response. A unique characteristic of M. ulcerans, is the fact that organisms secrete a putative toxin which is responsible for the distinctive features of disease. We have identified several biological properties of this toxin. These include the ability to cause cell cycle arrest, as well as the ability to profoundly alter the cytokine profile of infected cells. Using a partially purified toxic extract we have made the unexpected discovery that the toxic activity is associated with a polar lipid. In collaboration with Patrick Brennan's laboratory, the structure of the toxin is being elucidated.